Extracellular circulating miRNAs as stress-related signature to search and rescue dogs

Our research explores serum extracellular circulating miRNAs (ecmiRNAs) involved in dog stress response immediately after the search and rescue (SAR) of missing people. The experimental plan considers four arduous SAR simulations. The SAR dogs are trained by the Alpine School of the Military Force of Guardia di Finanza (Passo Rolle, Italy). The First SAR Trial analyzed dog serum samples at rest time (T0), and immediately after SAR performance (T1) using the miRNome-wide screening next-generation sequencing (NGS). T1 versus T0 NGS results revealed a different expression level of let-7a and let-7f. Subsequently, in a large sample size including: 1st (n = 6), 2nd (n = 6), 3rd (n = 6), and 4th (n = 4) trials, let-7a and let-7f were validated by qPCR. Bioinformatics analysis with TarBase (v.8) and the Diana-mirPath (v.3) revealed a functional role of let-7a and let-7f in the p53 pathway to restore cellular homeostasis. Let-7a and let-7f, highly expressed at T1, could stop MDMs-p53 inhibition inducing the p53 increase in level. In addition, let-7a and let-7f, via p53 post-transcriptional regulation, buffers p53 transcription spikes. During SAR stress, the possibility of p53 preconditioning could explain the phenomenon of “stress hardening” where the tolerance of particular stress increases after preconditioning.

The table shows the distribution and characteristics of the SAR dogs included in the trial. The dog identification (ID) is shown with a letter, and in parenthesis is indicated if male (M) or female (F).

SM 2. Hemolysis assessment during sample preparations
A significant source of variation in serum comes from contamination from cellular-derived ecmiRNA resulting from hemolysis. In serum samples, the level of hemolysis was measured using three methods. The first method (used in all processed serum samples) to assess hemolysis was a simple visual inspection of serum samples for pink discoloration, indicating free hemoglobin against a white background (carried out in the veterinarian field in all serum samples). The second method was used only for the samples processed with NGS (NGS SAR Trial samples) to monitor hemolysis and assessed the data from the red blood cell-specific miR-451a and the stable miR-23a determining the ratio miR-451a to miR-23a-3p (∆Cq; miR-23a-3p-miR-451a). The ∆Cq levels lower than 5 in serum represents non-hemolyzed samples. If the ∆Cq is close to or higher than 5, there is an increased risk of hemolysis. In all processed serum samples as a third method, recommended by QIAGEN for nonhuman serum samples, we evaluated hemoglobin concentration by the optical density at 414 nm (absorbance peak of free hemoglobin) using a NanoDrop 1000 spectrophotometer (THERMO SCIENTIFIC, Scoresby, Victoria, Australia). Samples were classified as hemolyzed if the oxyhemoglobin absorbance at 414 nm exceeded a value of 0.2 (third method). Serum samples (n = 22) hemolysis assessment, at the absorbance of 414 nm, showed a mean of 0.083, a standard deviation of 0.013, a minimum value of 0.068, and a maximum value of 0.12.

SM 3. RNA extraction and Spike-in for qPCR validations
Extraction was started by introducing 1 µL of spike-in mix*. The reagents and extraction method were provided by the MiRNeasy Serum/Plasma Extraction Kit (QIAGEN CLC bio, Aarhus, Denmark). At the end of extraction, RNA was eluted with 20 μl RNase-free water and centrifuged for 1 min at maximum speed. RNA integrity was assessed using RNA 2000 and Small RNA Chips on a Bioanalyzer (AGILENT TECHNOLOGIES, CA, USA).
* Only for qPCR sample validations. UniSp2, UniSp4, UniSp5 are added only in samples validated for qPCR, while a mixture of 52 spike-in, QIAseq miRNA Library QC Spike-Ins solution was added to serum samples processed for NGS (procedure described in MS 5). Before starting the RNA isolation procedure for qPCR validations, the UniSp2, UniSp4, UniSp5 spike-in mixture was resuspended as described by the manufacturer. After qPCR, the Ct value obtained permitted the control for varying RNA purification yields. In qPCR, UniSp5 was not detected because it corresponds to weakly expressed microRNAs.

SM 4. NGS serum miRNA Spike-in
Before starting the RNA isolation procedure are added 1 μL of QIAseq miRNA Library QC Spike-Ins solution for 400-500 μL serum as suggested in QIAseq miRNA Library QC PCR QIAGEN handbook. 52 QIAseq miRNA Library QC Spike-ins mix was used to assess the technical reproducibility and linearity of the mapped NGS reads. Mix the reaction thoroughly, dispense 10 μl from each well into qPCR plates, and spin the plate briefly. PCR cycling conditions included 15 min at 95 °C for enzyme PCR heat activation (at ambient temperature, the DNA Polymerase is kept inactive by antibody until the initial heat activation step); followed by 40 cycles of amplification: 15 sec 94 °C for denaturing double-stranded DNA, 15 sec 95 °C for annealing, and 15 sec 70 °C extension steps. Data acquisition should be performed during the annealing/extension step. Melting curve analysis 60-95 °C was performed to assess amplification specificity. The results interpreting spike-ins were performed referring to Qiagen suggestions. The PCR amplification efficiency was determined using the standard curve slope (efficiency = 10(−1/slope)−1). The slope of these graphs was utilized to determine the amplification efficiency. The PCR conditions were optimized to generate >95% PCR efficiency. Only reactions from 95 to 100% efficiency were included in the subsequent analysis.

SM 7. Spike-ins Quality control
The purpose of the RNA spike-in controls is to monitor the technical quality of RNA isolation and cDNA synthesis and to check PCR inhibitors in the sample. Spike-ins were amplified through PCR with relative forward and reverse primer mix. Following PCR, wells detecting the RNA spike-ins are compared, and outlier samples may be identified and considered for exclusion from further analysis. High variance >2 -3 Cq difference within a dataset for a given spike-in reflects high variance in RNA yields or potential sporadic RNase contamination. Wells detecting spike-in UniSp6 were compared, and outlier samples (Unisp6 Cq > 30) were excluded from data analysis. UniSp2 is present at a concentration 100-fold higher than UniSp4. Therefore, UniSp2 should amplify at the level of very abundant miRNAs; UniSp4 should amplify approximately 6.6 cycles later than UniSp2.